Trapping X‐ray Radiation Damage from Homolytic Se−C Bond Cleavage in BnSeSeBn Crystals (Bn=benzyl, CH2C6H5)

Abstract Irradiation of dibenzyl diselenide BnSeSeBn with X‐ray or UV‐light cleaves the Se−C and the Se−Se bonds, inducing stable and metastable radical states. They are inevitably important to all natural and life sciences. Structural changes due to X‐ray‐induced Se−C bond‐cleavage could be pin‐pointed in various high‐resolution X‐ray diffraction experiments for the first time. Extended DFT methods were applied to characterize the solid‐state structure and support the refinement of the observed residuals as contributions from the BnSeSe⋅ radical species. The X‐ray or UV‐irradiated crystalline samples of BnSeSeBn were characterized by solid‐state EPR. This paper provides insight that in the course of X‐ray structure analysis of selenium compounds not only organo‐selenide radicals like RSe⋅ may occur, but also organo diselenide BnSeSe⋅ radicals and organic radicals R⋅ are generated, particularly important to know in structural biology.

.717 (2) 13.7123 (18) 13.7199 (11) 13.7247 (7) 13.7299 (6) b [Å] 8.0012 (6) 8.0127 (13) 8.0051 (11) 8.0119 (6) 8.0053 (4) 8.0135 (4) c [Å] 11.4769(8) 11.4944 (18) 11.4849 (15) 11.4766 (9) 11.4831 (6) 11.4875(5) β [°] 99.293 (2) 99.303 (7) 99.260 (5) 99.301 (2) 99.2776 (16) [17] . Datasets C and D were collected with this detector. It provides a pixel wise adaptable energy-discrimination photon detection. By setting this threshold to a value above the selenium emission line at 12.7 KeV, the elevated background by X-ray fluorescence shown in the left image is effectively suppressed, leading to a very low background shown in the right image. The threshold was set to 15.5 keV for C and 13.0 keV for D; Bottom: four images with patterns that suggest diffuse scattering. Remarkably, this phenomenon occured right at the beginning of the data collection and did not aggravate.  Parameters were added stepwise to the refinement. Starting with multipole parameters, anisotropic motion and positional parameters, the refinement was extended by the application of anharmonic motion and the refinement of all selenium multipole parameters. In the penultimate step, κ' parameters were refined, followed by a refinement of all prior introduced parameters. Hydrogen positional parameter were refined only against low-resolution data up to 0.5 sin(θ)/λ, while constrained to neutron distances.  The refinement strategy is analogous to the refinement strategy in XD (Table S9), but slightly shortened due to program restrictions. Additionally, 32-poles at selenium were introduced in the penultimate and 64-poles in the ultimate step of the refinement.  Positive contours are plotted with green lines and negative contours are plotted with red lines. The graphics were created with MoleCoolQT [18] Table S11. Consistency check for the refinement of Gram Charlier parameters of 3 rd and 4 th order at Se(1) in XD. Analysis of the Probability Density Function (PDF). Extreme displacements in the map from the equilibrium position. ∆X/Y/Z = -0.80 to 0.80Å. For datasets B to F, the minimum PDF value and integrated volume for negative probability are reasonably low, while for dataset A, the values are slightly elevated. In order to retain comparability, anharmonic motion was refined anyway.  Table S12.
For an anharmonic refinement, Kuhs's rule [12] should be fulfilled. However, this rule seems to be too strict for heavier atoms. [19] Therefore, anharmonic motion of Se(1) was refined for all datasets up to the fourth order although the used resolution is only sin( )/ = 1.  Table S13.
Significance check for the refinement of Gram Charlier parameters of 3 rd and 4 th order at Se(1) in XD. Gram-Charlier parameter divided by their errorin order to be significant, values need to be larger than 3.   [13][14][15] for the XD refinement of dataset A.

Fig. S9:
Fractal dimension plot [16] for the XD refinement of dataset A.

Fig. S13:
Fractal dimension plot [16] for the XD refinement of dataset B.

Fig. S17:
Fractal dimension plot [16] for the XD refinement of dataset C.

Fig. S29:
Fractal dimension plot [16] for the XD refinement of dataset F.

Computational Details
All calculations were carried out with the additive crystal QM/MM model (AC-QM/MM) [21] implemented in a modified version of the Chemshell 3.5.0 [22] program. The built-in DL-POLY module was used for the MM part, the ORCA 3.0.3 [23] program package was used for the QM part. QM calculations employed the B3LYP functional [24] in conjunction with the def2-TZVP basis set [25] . D3 dispersion corrections [26] to the functional were applied with Becke-Johnson damping [27] . The RIJ approximation to the Coulomb part [28] and COSX approximation [29] to the exchange part were used throughout all calculations with the def2-TZVP/J auxiliary basis set [30] .
AC-QM/MM calculations were carried out at the one-body level, since exemplary two-body results for the main structure 1, based on dataset C, provided essentially no changes in the geometry. A distance cutoff of 40 bohr was used to generate the cluster. NPA charges [31] were used for the electrostatic embedding, Lennard-Jones parameters from the Universal Force Field [32] (selenium only) and Charmm Generalised Force Field [33] (all other elements) for the further embedding.
Within the AC-QM/MM framework, the energy expression was modified to represent a disordered molecule in an ordered environment. The optimization is not carried out with respect to the cohesive energy Ecoh, as defined in the AC-QM/MM model, but with respect to the embedded energy Eemb. The former is defined only for an unperturbed crystal, representing the stabilization of a molecule in the crystal compared to a gas phase molecule. It cannot be applied for this problem, as the underlying theory requires all molecules in the crystal to be the same. The embedded energy is apt to describe the relative stabilization of the disordered molecule in the frozen environment and allows for a reasonable optimization, but its absolute values do not have a physical meaning.
AC-QM/MM calculations were performed in two steps. Firstly, the crystal structure of 1, based on dataset C, was optimized without restrictions, i.e. both the geometry and cell parameters were relaxed. The resulting structure was used as the foundation for further calculations. The environment was frozen: Geometries and embedding potentials were kept fixed at the values of this calculation.
For subsequent calculations, the QM molecule in the cluster was optimized in the frozen environment, starting from three different geometries and simulating six different electronic states. As starting geometry 1, the experimental molecular structure was used unchanged. For 2, the Se atoms were moved to the mean residual density peak positions of A to F. For 3 the Se-Se distance was elongated to 2.894 Å, the mean value of intra-and intermolecular Se-Se distance.
The electronic states taken into consideration were the singlet ground state (S0) and first excited state (S1), the lowest triplet state (T0), and a broken symmetry state (BS). Furthermore, a singly charged cation and anion in their doublet ground states (D0) were considered. The starting structure with Se atoms at the residual density peak positions was considered the best experimental estimation of the structure of 2. In Table S14, the resulting geometries are compared to the experimental structure of 1 (Dataset C) and the starting conformation of 2 with Se at the residual density peak positions. Optimized structures of (BnSe)2 1, BnSe-Se* *Bn 2 and BnSe* *SeBn 3 are labelled according to their electronic state, e.g. 1(S0).
For the 1(S0) and 1(BS) state, the starting geometry is retained. For 1(S1) and 1(T0) an elongation of the Se-Se bond is observed, resembling 3. The cation 1(D0 + ) leads to a significant torsion of the C-Se-Se-C dihedral angle, slightly smaller Se-Se and slightly larger C-Se distances do not resemble the experimental expectation. For the anion 1(D0 -), an elongated Se-Se bond is observed, but quite similar residual geometry parameters.
Starting from the elongated Se-Se structure 3, the optimizations 3(S0) to 3(D0 -) yield very similar, virtually unchanged structure for all electronic states with only negligible differences in geometry.
The optimization starting from the experimental residual density peak positions (2) provide interesting results. 2(S0) and 2(BS) relax to the ground-state geometry like 1(S0), as expected. For 2(S1) no energy minimum could be determined. A crossing with the S0 energy surface was observed, thus indicating that at least in that structural region no energy minimum of the first excited singlet state is accessible. 2(T0) state did not relax to a 3 akin structure, as it had been the case for both other starting geometries. Although the starting structure was not retained, the general agreement of key geometry parameters between the experimental residual density peak positions and 2(T0) is quite reasonable. The Se-Se distance is notably longer (2.243 Å) compared to the residual density peak distance (2.170 Å) while one C-Se bond is shorter (2.004 Å) than in the experimental estimation (2.167 Å). However, the broken C-Se bond is very similar (3.132 Å) to the experimental estimation (3.155 Å) and one of the C-Se-Se angles is very close (100.8° to 93.3°). For the second angle (79.7°) the agreement to the experimental estimation (52.7°) is notably worse, as well for the C-Se-Se-C dihedral (109.2° to 126°). Nonetheless, it must be considered that in all parameters the results obtained from 2(T0) are not only in much better agreement than all other results, but also the deviations from experimental 1 as well as 1(S0) are in the direction of the residual density peaks. Hence, it is not considered a clear evidence of the experimental structure representing a triplet state, but it strongly supports the assumption.
EPR parameters were calculated for the optimized triplet structures. Parameters for 2(T0) and 3(T0) were found to be notably different, thus allowing for an assignment of the experimental EPR results. The EPR spectra were calculated from the theoretical g tensors as described in the respective EPR part of this document. Table S14. Selected geometry parameters of optimized structures for different starting structures (Init) optimized in different electronic states (State) and compared to the experimental parameters (EXP), as well as the residual density peak positions as Se-atoms (EXPQ).

Init
State

Residual Density Analysis Details
Model preparation In order to achieve multipole populations unbiased by experimental errors, theoretical scattering factors were calculated based on the optimized structure 1(S0) using DenProp [34] . The atom positions were retained and only the multipole parameters were refined against the data, using the same restraints and local symmetry as for datasets A to F. The deformation density (Fig. S34) reproduces the electron densities features as found in Fig. S33. The residual density map level is reasonably low and mainly shows spherical features around the selenium positions (Fig. S34). This feature is common for the multipole-description of heavy atoms from calculated data and originates from the core-polarization [35] .

Residual density determination
The determined multipole populations were applied to datasets A to F. Here, only atom positions, vibrational parameters and the scaling factor were refined against the experimental data, while the multipole populations were retained. The resulting residual density gives a good estimation of the differences between the theoretically expected and the experimentally determined density.

EPR Analysis Details
Clean samples of solid (BnSe)2 were freshly recrystallized from tetrahydrofuran, ground thoroughly and were placed in Ø=5mm EPR tubes. UV-irradiated samples were irradiated for 6 hours with a 150 W Hg(Xe) arc lamp with a housing and power supply from LOT-Quantum Design GmbH. IR irradiation was cut off by a water filter. The sample was kept in liquid nitrogen. X-ray irradiated samples were irradiated in the full 'pink' beam of a 1200 W Rigaku MicroMax 007 rotating anode (Cu Kα), while cooled to approximately 100 K in a cold gaseous N2 stream.
EPR spectra were collected within half an hour after irradiation, using a ELEXSYS CW-EPR spectrometer E500, equipped with a digital cryo-system ER 4131 VT at a microwave frequency of approximately 9.42 GHz, 1x10 -4 G modulation amplitude, 100 kHz modulation frequency, and a microwave power of 5-6 mW in a temperature range between 142 and 290 K. The samples were re-evaluated after 1-2 days and in the case of the Xray irradiated sample again after 30 days. The X-ray irradiated sample was re-evaluated after 60 days, using a Bruker E580 pulse EPR spectrometer operating at X band frequency for pulsed EPR measurements and a Bruker E500 ELEXYS CW-EPR spectrometer. Pulsed EPR measurements were recorded with a spin echo sequence using mw pulses of 20 and 40 ns for 90 and 180 degrees, respectively. The delays between first and second pulse were 420 ns (red spectrum) and 3080 ns (blue spectrum) to separate species with differing spin-spin-relaxation times. Spectra were recorded at 298 K at a microwave frequency of 9.60 GHz, acquiring two scans with 500 shot/point. CW EPR spectra were recorded at a mw frequency of 9.84 GHz, 0.2 G modulation amplitude, 100 kHz modulation frequency, and a mw power of 6-7 mW. Fig. S43B shows the EPR signal of a UV-irradiated sample, which was not fully submerged in liquid nitrogen during the irradiation. The red discoloration of the sample in that area and the otherwise not detectable signal at g = 2.9 brought us to the conclusion to amorphous selenium [36] in contrast to other contemporary studies [37] .

Fig. S46:
Detailed EPR signals of the main peaks of an X-ray irradiated sample and the same sample after 60 days.